Gene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry

The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits. SIGNIFICANCE STATEMENT The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.

[1]  Sachie K. Ogawa,et al.  Whole-Brain Mapping of Direct Inputs to Midbrain Dopamine Neurons , 2012, Neuron.

[2]  Allan R. Jones,et al.  A mesoscale connectome of the mouse brain , 2014, Nature.

[3]  Lisa E. Pomeranz,et al.  Molecular Biology of Pseudorabies Virus: Impact on Neurovirology and Veterinary Medicine , 2005, Microbiology and Molecular Biology Reviews.

[4]  David G Hendrickson,et al.  Differential analysis of gene regulation at transcript resolution with RNA-seq , 2012, Nature Biotechnology.

[5]  A. Bonci,et al.  Serotonergic versus nonserotonergic dorsal raphe projection neurons: differential participation in reward circuitry. , 2014, Cell reports.

[6]  Y. Smith,et al.  A cocaine-and-amphetamine-regulated-transcript peptide projection from the lateral hypothalamus to the ventral tegmental area , 2005, Neuroscience.

[7]  J. Friedman,et al.  Virus-Assisted Mapping of Neural Inputs to a Feeding Center in the Hypothalamus , 2001, Science.

[8]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[9]  L. Enquist,et al.  Transneuronal Circuit Analysis with Pseudorabies Viruses , 1999, Current protocols in neuroscience.

[10]  Talia N. Lerner,et al.  Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits , 2015, Cell.

[11]  K. Conzelmann,et al.  Melanocortin 3 Receptor Signaling in Midbrain Dopamine Neurons Increases the Motivation for Food Reward , 2016, Neuropsychopharmacology.

[12]  Kevin T. Beier,et al.  Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors , 2011, Proceedings of the National Academy of Sciences.

[13]  G. Cano,et al.  Dual viral transneuronal tracing of central autonomic circuits involved in the innervation of the two kidneys in rat , 2004, The Journal of comparative neurology.

[14]  Lynn W Enquist,et al.  Recent advances in the use of neurotropic viruses for circuit analysis , 2003, Current Opinion in Neurobiology.

[15]  L. Weaver,et al.  Simultaneous identification of two populations of sympathetic preganglionic neurons using recombinant herpes simplex virus type 1 expressing different reporter genes , 1997, Neuroscience.

[16]  Stephen A. Allsop,et al.  Decoding Neural Circuits that Control Compulsive Sucrose Seeking , 2015, Cell.

[17]  K. Deisseroth,et al.  Input-specific control of reward and aversion in the ventral tegmental area , 2012, Nature.

[18]  Toshio Iijima,et al.  Dual Transneuronal Tracing in the Rat Entorhinal-Hippocampal Circuit by Intracerebral Injection of Recombinant Rabies Virus Vectors , 2008, Front. Neuroanat..

[19]  Feilong He,et al.  Genome-Wide Transcriptional Profiling Reveals Two Distinct Outcomes in Central Nervous System Infections of Rabies Virus , 2016, Front. Microbiol..

[20]  A. Herbison,et al.  Definition of brainstem afferents to gonadotropin-releasing hormone neurons in the mouse using conditional viral tract tracing. , 2007, Endocrinology.

[21]  R. España,et al.  Hypocretin/Orexin Regulation of Dopamine Signaling and Cocaine Self-Administration Is Mediated Predominantly by Hypocretin Receptor 1 , 2014, ACS chemical neuroscience.

[22]  S. Geisler,et al.  Afferents of the ventral tegmental area in the rat‐anatomical substratum for integrative functions , 2005, The Journal of comparative neurology.

[23]  J. Schall,et al.  Visual and Motor Connectivity and the Distribution of Calcium-Binding Proteins in Macaque Frontal Eye Field: Implications for Saccade Target Selection , 2009, Front. Neuroanat..

[24]  David J. Anderson,et al.  Control of Stress-Induced Persistent Anxiety by an Extra-Amygdala Septohypothalamic Circuit , 2014, Cell.

[25]  P. Strick,et al.  Rabies as a transneuronal tracer of circuits in the central nervous system , 2000, Journal of Neuroscience Methods.

[26]  T. Hökfelt,et al.  Molecular diversity of corticotropin-releasing hormone mRNA-containing neurons in the hypothalamus. , 2017, The Journal of endocrinology.

[27]  M. Ekstrand,et al.  The alpha-herpesviruses: molecular pathfinders in nervous system circuits. , 2008, Trends in molecular medicine.

[28]  B. Banfield,et al.  Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications , 2003, Journal of Virology.

[29]  C. Rogel-Gaillard,et al.  Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection , 2008, BMC Genomics.

[30]  M. Ekstrand,et al.  Molecular characterization of neuronal cell types based on patterns of projection with Retro-TRAP , 2015, Nature Protocols.

[31]  Andrew D Huberman,et al.  Transsynaptic Tracing with Vesicular Stomatitis Virus Reveals Novel Retinal Circuitry , 2013, The Journal of Neuroscience.

[32]  Naoshige Uchida,et al.  Organization of monosynaptic inputs to the serotonin and dopamine neuromodulatory systems. , 2014, Cell reports.

[33]  J. Crawley,et al.  Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons , 2002, Journal of neurochemistry.

[34]  István Prazsák,et al.  Characterization of pseudorabies virus transcriptome by Illumina sequencing , 2015, BMC Microbiology.

[35]  L. Enquist,et al.  A self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  George Paxinos,et al.  The Mouse Brain in Stereotaxic Coordinates , 2001 .

[37]  L. Enquist,et al.  Transcriptome Signature of Virulent and Attenuated Pseudorabies Virus-Infected Rodent Brain , 2006, Journal of Virology.

[38]  S. Ikemoto Dopamine reward circuitry: Two projection systems from the ventral midbrain to the nucleus accumbens–olfactory tubercle complex , 2007, Brain Research Reviews.

[39]  Liqun Luo,et al.  Diversity of Transgenic Mouse Models for Selective Targeting of Midbrain Dopamine Neurons , 2015, Neuron.

[40]  Z. Knight,et al.  Molecular Profiling of Neurons Based on Connectivity , 2014, Cell.

[41]  B. Waterhouse,et al.  Functional organization of the dorsal raphe efferent system with special consideration of nitrergic cell groups , 2011, Journal of Chemical Neuroanatomy.

[42]  Lynn W Enquist,et al.  Inputs to serotonergic neurons revealed by conditional viral transneuronal tracing , 2009, The Journal of comparative neurology.

[43]  Liqun Luo,et al.  Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping , 2015, Cell.

[44]  L. Enquist,et al.  Exploiting circuit-specific spread of pseudorabies virus in the central nervous system: insights to pathogenesis and circuit tracers. , 2002, The Journal of infectious diseases.

[45]  Ana I. Domingos,et al.  Hypothalamic melanin concentrating hormone neurons communicate the nutrient value of sugar , 2013, eLife.

[46]  A. Loewy,et al.  Central Command Neurons of the Sympathetic Nervous System: Basis of the Fight-or-Flight Response , 1995, Science.

[47]  Ian R. Wickersham,et al.  Retrograde neuronal tracing with a deletion-mutant rabies virus , 2007, Nature Methods.

[48]  Toshio Iijima,et al.  Untangling Neural Networks with Dual Retrograde Transsynaptic Viral Infection , 2009, Front. Neurosci..

[49]  Eva Lindqvist,et al.  Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons , 2007, Proceedings of the National Academy of Sciences.

[50]  L. Enquist,et al.  Transcriptional Response of a Common Permissive Cell Type to Infection by Two Diverse Alphaherpesviruses , 2004, Journal of Virology.

[51]  L. Enquist,et al.  Olfactory Inputs to Hypothalamic Neurons Controlling Reproduction and Fertility , 2005, Cell.

[52]  Kevin T. Beier,et al.  Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms , 2015, The Journal of comparative neurology.

[53]  Ian R. Wickersham,et al.  Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons , 2007, Neuron.

[54]  Yaping Zhang,et al.  Cellular responses to HSV-1 infection are linked to specific types of alterations in the host transcriptome , 2016, Scientific Reports.

[55]  G. Ugolini,et al.  Rabies virus as a transneuronal tracer of neuronal connections. , 2011, Advances in virus research.

[56]  L. Enquist,et al.  Neuroinvasiveness of pseudorabies virus injected intracerebrally is dependent on viral concentration and terminal field density , 1999, The Journal of comparative neurology.

[57]  E. ten Dam,et al.  The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences. , 2001, The Journal of general virology.

[58]  L. Enquist,et al.  Intravitreal Injection of the Attenuated Pseudorabies Virus PRV Bartha Results in Infection of the Hamster Suprachiasmatic Nucleus Only by Retrograde Transsynaptic Transport via Autonomic Circuits , 2002, The Journal of Neuroscience.

[59]  Moriah L. Szpara,et al.  A Common Neuronal Response to Alphaherpesvirus Infection , 2010, Journal of Neuroimmune Pharmacology.

[60]  Liqun Luo,et al.  Viral-genetic tracing of the input–output organization of a central norepinephrine circuit , 2015, Nature.

[61]  N. DeLuca,et al.  Transcription of the Herpes Simplex Virus 1 Genome during Productive and Quiescent Infection of Neuronal and Nonneuronal Cells , 2014, Journal of Virology.

[62]  David J. Anderson,et al.  A Cre-Dependent, Anterograde Transsynaptic Viral Tracer for Mapping Output Pathways of Genetically Marked Neurons , 2011, Neuron.

[63]  Yaron Lipman,et al.  Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes , 2010, Nature communications.

[64]  F. Fujiyama,et al.  Vesicular glutamate transporter 3‐expressing nonserotonergic projection neurons constitute a subregion in the rat midbrain raphe nuclei , 2010, The Journal of comparative neurology.

[65]  Nancy A. Jenkins,et al.  Simple and highly efficient BAC recombineering using galK selection , 2005, Nucleic acids research.

[66]  Floris G. Wouterlood,et al.  A half century of experimental neuroanatomical tracing , 2011, Journal of Chemical Neuroanatomy.

[67]  Allan R. Jones,et al.  Genome-wide atlas of gene expression in the adult mouse brain , 2007, Nature.

[68]  Lynn W. Enquist,et al.  A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits , 2011, PloS one.

[69]  K. Deisseroth,et al.  Rapid regulation of depression-related behaviors by control of midbrain dopamine neurons , 2012, Nature.

[70]  Juancarlos Chan,et al.  Gene Ontology Consortium: going forward , 2014, Nucleic Acids Res..

[71]  Minmin Luo,et al.  Reward processing by the dorsal raphe nucleus: 5-HT and beyond , 2015, Learning & memory.

[72]  G. Gentry,et al.  Inhibition of herpes simplex virus replication by araT. , 1975, Virology.